Indolent CD4+ CAR T-Cell Lymphoma after Cilta-cel CAR T-Cell Therapy.

Indolent CD4+ cytotoxic chimeric antigen receptor (CAR) T-cell lymphoma involving the small intestine was diagnosed in a patient who had previously received ciltacabtagene autoleucel (cilta-cel) CAR T-cell therapy for treatment of myeloma. Targeted messenger RNA sequencing revealed the presence of CAR gene product in tumor cells. Whole-genome sequencing of samples of tumor and peripheral blood identified a single lentiviral insertion site within the second intron of the SSU72 gene. In addition, numerous genetic alterations that may have contributed to malignant transformation were identified in the tumor sample. (Funded by MedStar Georgetown University Hospital.).

Anticancer Drugs of Lysine Specific Histone Demethylase-1 (LSD1) Display Variable Inhibition on Nucleosome Substrates.

Lysine specific demethylase-1 (LSD1) serves as a regulator of transcription and represents a promising epigenetic target for anticancer treatment. LSD1 inhibitors are in clinical trials for the treatment of Ewing's sarcoma (EWS), acute myeloid leukemia, and small cell lung cancer, and the development of robust inhibitors requires accurate methods for probing demethylation, potency, and selectivity. Here, the inhibition kinetics on the H3K4me2 peptide and nucleosome substrates was examined, comparing the rates of demethylation in the presence of reversible [CC-90011 (PD) and SP-2577 (SD)] and irreversible [ORY-1001 (ID) and tranylcypromine (TCP)] inhibitors. Inhibitors were also subject to viability studies in three human cell lines and Western blot assays to monitor H3K4me2 nucleosome levels in EWS (TC-32) cells, enabling a correlation of drug potency, inhibition in vitro, and cell-based studies. For example, SP-2577, a drug in clinical trials for EWS, inhibits activity on small peptide substrates (Ki = 60 ± 20 nM) using an indirect coupled assay but does not inhibit demethylation on H3K4me2 peptides or nucleosomes using direct Western blot approaches. In addition, the drug has no effect on H3K4me2 levels in TC-32 cells. These data show that SP-2577 is not an LSD1 enzyme inhibitor, although the drug may function independent of demethylation due to its cytotoxic selectivity in TC-32 cells. Taken together, this work highlights the pitfalls of using coupled assays to ascribe a drug's mode of action, emphasizes the use of physiologically relevant substrates in epigenetic drug targeting strategies, and provides insight into the development of substrate-selective inhibitors of LSD1.

EWS-FLI1 modulated alternative splicing of ARID1A reveals novel oncogenic function through the BAF complex.

Connections between epigenetic reprogramming and transcription or splicing create novel mechanistic networks that can be targeted with tailored therapies. Multiple subunits of the chromatin remodeling BAF complex, including ARID1A, play a role in oncogenesis, either as tumor suppressors or oncogenes. Recent work demonstrated that EWS-FLI1, the oncogenic driver of Ewing sarcoma (ES), plays a role in chromatin regulation through interactions with the BAF complex. However, the specific BAF subunits that interact with EWS-FLI1 and the precise role of the BAF complex in ES oncogenesis remain unknown. In addition to regulating transcription, EWS-FLI1 also alters the splicing of many mRNA isoforms, but the role of splicing modulation in ES oncogenesis is not well understood. We have identified a direct connection between the EWS-FLI1 protein and ARID1A isoform protein variant ARID1A-L. We demonstrate here that ARID1A-L is critical for ES maintenance and supports oncogenic transformation. We further report a novel feed-forward cycle in which EWS-FLI1 leads to preferential splicing of ARID1A-L, promoting ES growth, and ARID1A-L reciprocally promotes EWS-FLI1 protein stability. Dissecting this interaction may lead to improved cancer-specific drug targeting.

Protein Phosphatase 1-Targeting Small-Molecule C31 Inhibits Ebola Virus Replication.

Background: Ebola virus (EBOV) infection causes severe hemorrhagic fever. EBOV transcription is controlled by host protein phosphatase 1 (PP1), which dephosphorylates VP30 protein. We previously developed 1E7-03, a compound targeting a noncatalytic site of PP1 that induced VP30 phosphorylation and inhibited EBOV transcription. Here, we attempted to further improve 1E7-03, which was not stable in murine serum. Results: High-throughput screening with EBOV-green fluorescent protein was conducted on 72 1E7-03 analogs and identified 6 best inhibitory and the least toxic compounds. A parallel in silico screening of compounds from the ZINC database by docking to PP1 identified the best-binding compound C31, which was also present among the top 6 compounds found in the viral screen. C31 showed the best EBOV inhibitory activity among the top 6 compounds and also inhibited EBOV minigenome. C31 bound to the PP1 C-terminal groove in vitro and increased VP30 phosphorylation in cultured cells. C31 demonstrated improved stability in mouse plasma and cell permeability, compared with 1E7-03. It was also detected for 24 hours after injection in mice. Conclusion: C31 represents a novel PP1-targeting EBOV inhibitor with improved pharmacological properties that can be further evaluated for future antifiloviral therapy.

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing.

The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.

Ezrin Inhibition Up-regulates Stress Response Gene Expression.

Ezrin is a member of the ERM (ezrin/radixin/moesin) family of proteins that links cortical cytoskeleton to the plasma membrane. High expression of ezrin correlates with poor prognosis and metastasis in osteosarcoma. In this study, to uncover specific cellular responses evoked by ezrin inhibition that can be used as a specific pharmacodynamic marker(s), we profiled global gene expression in osteosarcoma cells after treatment with small molecule ezrin inhibitors, NSC305787 and NSC668394. We identified and validated several up-regulated integrated stress response genes including PTGS2, ATF3, DDIT3, DDIT4, TRIB3, and ATF4 as novel ezrin-regulated transcripts. Analysis of transcriptional response in skin and peripheral blood mononuclear cells from NSC305787-treated mice compared with a control group revealed that, among those genes, the stress gene DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of ezrin inhibitor compound activity. In addition, we validated the anti-metastatic effects of NSC305787 in reducing the incidence of lung metastasis in a genetically engineered mouse model of osteosarcoma and evaluated the pharmacokinetics of NSC305787 and NSC668394 in mice. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive protein, has important functions in regulating gene expression that may result in down-regulation of stress response genes.

Interaction Between HIV-1 Nef and Calnexin: From Modeling to Small Molecule Inhibitors Reversing HIV-Induced Lipid Accumulation.

OBJECTIVE: HIV-infected patients are at an increased risk of developing atherosclerosis, in part because of downmodulation and functional impairment of ATP-binding cassette A1 (ABCA1) cholesterol transporter by the HIV-1 protein Nef. The mechanism of this effect involves Nef interacting with an ER chaperone calnexin and disrupting calnexin binding to ABCA1, leading to ABCA1 retention in ER, its degradation and resulting suppression of cholesterol efflux. However, molecular details of Nef-calnexin interaction remained unknown, limiting the translational impact of this finding. APPROACH AND RESULTS: Here, we used molecular modeling and mutagenesis to characterize Nef-calnexin interaction and to identify small molecule compounds that could block it. We demonstrated that the interaction between Nef and calnexin is direct and can be reconstituted using recombinant proteins in vitro with a binding affinity of 89.1 nmol/L measured by surface plasmon resonance. The cytoplasmic tail of calnexin is essential and sufficient for interaction with Nef, and binds Nef with an affinity of 9.4 nmol/L. Replacing lysine residues in positions 4 and 7 of Nef with alanines abrogates Nef-calnexin interaction, prevents ABCA1 downregulation by Nef, and preserves cholesterol efflux from HIV-infected cells. Through virtual screening of the National Cancer Institute library of compounds, we identified a compound, 1[(7-oxo-7H-benz[de]anthracene-3-yl)amino]anthraquinone, which blocked Nef-calnexin interaction, partially restored ABCA1 activity in HIV-infected cells, and reduced foam cell formation in a culture of HIV-infected macrophages. CONCLUSION: This study identifies potential targets that can be exploited to block the pathogenic effect of HIV infection on cholesterol metabolism and prevent atherosclerosis in HIV-infected subjects.

RNA helicase A activity is inhibited by oncogenic transcription factor EWS-FLI1.

RNA helicases impact RNA structure and metabolism from transcription through translation, in part through protein interactions with transcription factors. However, there is limited knowledge on the role of transcription factor influence upon helicase activity. RNA helicase A (RHA) is a DExH-box RNA helicase that plays multiple roles in cellular biology, some functions requiring its activity as a helicase while others as a protein scaffold. The oncogenic transcription factor EWS-FLI1 requires RHA to enable Ewing sarcoma (ES) oncogenesis and growth; a small molecule, YK-4-279 disrupts this complex in cells. Our current study investigates the effect of EWS-FLI1 upon RHA helicase activity. We found that EWS-FLI1 reduces RHA helicase activity in a dose-dependent manner without affecting intrinsic ATPase activity; however, the RHA kinetics indicated a complex model. Using separated enantiomers, only (S)-YK-4-279 reverses the EWS-FLI1 inhibition of RHA helicase activity. We report a novel RNA binding property of EWS-FLI1 leading us to discover that YK-4-279 inhibition of RHA binding to EWS-FLI1 altered the RNA binding profile of both proteins. We conclude that EWS-FLI1 modulates RHA helicase activity causing changes in overall transcriptome processing. These findings could lead to both enhanced understanding of oncogenesis and provide targets for therapy.

Germ line variants of human N-methylpurine DNA glycosylase show impaired DNA repair activity and facilitate 1,N6-ethenoadenine-induced mutations.

Human N-methylpurine DNA glycosylase (hMPG) initiates base excision repair of a number of structurally diverse purine bases including 1,N(6)-ethenoadenine, hypoxanthine, and alkylation adducts in DNA. Genetic studies discovered at least eight validated non-synonymous single nucleotide polymorphisms (nsSNPs) of the hMPG gene in human populations that result in specific single amino acid substitutions. In this study, we tested the functional consequences of these nsSNPs of hMPG. Our results showed that two specific arginine residues, Arg-141 and Arg-120, are important for the activity of hMPG as the germ line variants R120C and R141Q had reduced enzymatic activity in vitro as well as in mammalian cells. Expression of these two variants in mammalian cells lacking endogenous MPG also showed an increase in mutations and sensitivity to an alkylating agent compared with the WT hMPG. Real time binding experiments by surface plasmon resonance spectroscopy suggested that these variants have substantial reduction in the equilibrium dissociation constant of binding (KD) of hMPG toward 1,N(6)-ethenoadenine-containing oligonucleotide (ϵA-DNA). Pre-steady-state kinetic studies showed that the substitutions at arginine residues affected the turnover of the enzyme significantly under multiple turnover condition. Surface plasmon resonance spectroscopy further showed that both variants had significantly decreased nonspecific (undamaged) DNA binding. Molecular modeling suggested that R141Q substitution may have resulted in a direct loss of the salt bridge between ϵA-DNA and hMPG, whereas R120C substitution redistributed, at a distance, the interactions among residues in the catalytic pocket. Together our results suggest that individuals carrying R120C and R141Q MPG variants may be at risk for genomic instability and associated diseases as a consequence.

Identification of a binding site of the human immunodeficiency virus envelope protein gp120 to neuronal-specific tubulin.

Human immunodeficiency virus-1 (HIV) promotes synaptic simplification and neuronal apoptosis, and causes neurological impairments termed HIV-associated neurological disorders. HIV-associated neurotoxicity may be brought about by acute and chronic mechanisms that still remain to be fully characterized. The HIV envelope glycoprotein gp120 causes neuronal degeneration similar to that observed in HIV-associated neurocognitive disorders subjects. This study was undertaken to discover novel mechanisms of gp120 neurotoxicity that could explain how the envelope protein promotes neurite pruning. Gp120 has been shown to associate with various intracellular organelles as well as microtubules in neurons. We then analyzed lysates of neurons exposed to gp120 with liquid chromatography mass spectrometry for potential protein interactors. We found that one of the proteins interacting with gp120 is tubulin ß-3 (TUBB3), a major component of neuronal microtubules. We then tested the hypothesis that gp120 binds to neuronal microtubules. Using surface plasmon resonance, we confirmed that gp120 binds with high affinity to neuronal-specific TUBB3. We have also identified the binding site of gp120 to TUBB3. We then designed a small peptide (Helix-A) that displaced gp120 from binding to TUBB3. To determine whether this peptide could prevent gp120-mediated neurotoxicity, we cross-linked Helix-A to mesoporous silica nanoparticles (Helix-A nano) to enhance the intracellular delivery of the peptide. We then tested the neuroprotective property of Helix-A nano against three strains of gp120 in rat cortical neurons. Helix-A nano prevented gp120-mediated neurite simplification as well as neuronal loss. These data propose that gp120 binding to TUBB3 could be another mechanism of gp120 neurotoxicity. We propose a novel direct mechanism of human immunodeficiency virus neurotoxicity. Our data show that the viral protein gp120 binds to neuronal specific tubulin ß-3 and blocks microtubule transport. Displacing gp120 from binding to tubulin by a small peptide prevents gp120-mediated neuronal loss. Our study reveals a novel target for developing adjunct therapies against viral infection that promotes neurocognitive disorders.

Characterizing the molecular features of ERG-positive tumors in primary and castration resistant prostate cancer.

BACKGROUND: The TMPRSS2-ERG gene fusion is detected in approximately half of primary prostate cancers (PCa) yet the prognostic significance remains unclear. We hypothesized that ERG promotes the expression of common genes in primary PCa and metastatic castration-resistant PCa (CRPC), with the objective of identifying ERG-associated pathways, which may promote the transition from primary PCa to CRPC. METHODS: We constructed tissue microarrays (TMA) from 127 radical prostatectomy specimens, 20 LuCaP patient-derived xenografts (PDX), and 152 CRPC metastases obtained immediately at time of death. Nuclear ERG was assessed by immunohistochemistry (IHC). To characterize the molecular features of ERG-expressing PCa, a subset of IHC confirmed ERG+ or ERG- specimens including 11 radical prostatectomies, 20 LuCaP PDXs, and 45 CRPC metastases underwent gene expression analysis. Genes were ranked based on expression in primary PCa and CRPC. Common genes of interest were targeted for IHC analysis and expression compared with biochemical recurrence (BCR) status. RESULTS: IHC revealed that 43% of primary PCa, 35% of the LuCaP PDXs, and 18% of the CRPC metastases were ERG+ (12 of 48 patients [25%] had at least one ERG+ metastasis). Based on gene expression data and previous literature, two proteins involved in calcium signaling (NCALD, CACNA1D), a protein involved in inflammation (HLA-DMB), CD3 positive immune cells, and a novel ERG-associated protein, DCLK1 were evaluated in primary PCa and CRPC metastases. In ERG+ primary PCa, a weak association was seen with NCALD and CACNA1D protein expression. HLA-DMB association with ERG was decreased and CD3 cell number association with ERG was changed from positive to negative in CRPC metastases compared to primary PCa. DCLK1 was upregulated at the protein level in unpaired ERG+ primary PCa and CRPC metastases (P = 0.0013 and P < 0.0001, respectively). In primary PCa, ERG status or expression of targeted proteins was not associated with BCR-free survival. However, for primary PCa, ERG+DCLK1+ patients exhibited shorter time to BCR (P = 0.06) compared with ERG+DCLK1- patients. CONCLUSIONS: This study examined ERG expression in primary PCa and CRPC. We have identified altered levels of inflammatory mediators associated with ERG expression. We determined expression of DCLK1 correlates with ERG expression and may play a role in primary PCa progression to metastatic CPRC. Prostate 76:810-822, 2016. © 2016 Wiley Periodicals, Inc.

Reactivation of latent HIV-1 provirus via targeting protein phosphatase-1.

BACKGROUND: HIV-1 escapes antiretroviral drugs by integrating into the host DNA and forming a latent transcriptionally silent HIV-1 provirus. This provirus presents the major hurdle in HIV-1 eradication and cure. Transcriptional activation, which is prerequisite for reactivation and the eradication of latent proviruses, is impaired in latently infected T cells due to the lack of host transcription factors, primarily NF-κB and P-TEFb (CDK9/cyclin T1). We and others previously showed that protein phosphatase-1 (PP1) regulates HIV-1 transcription by modulating CDK9 phosphorylation. Recently we have developed a panel of small molecular compounds targeting a non-catalytic site of PP1. RESULTS: Here we generated a new class of sulfonamide-containing compounds that activated HIV-1 in acute and latently infected cells. Among the tested molecules, a small molecule activator of PP1 (SMAPP1) induced both HIV-1 replication and reactivation of latent HIV-1 in chronically infected cultured and primary cells. In vitro, SMAPP1 interacted with PP1 and increased PP1 activity toward a recombinant substrate. Treatment with SMAPP1 increased phosphorylation of CDK9's Ser90 and Thr186 residues, but not Ser175. Proteomic analysis showed upregulation of P-TEFb and PP1 related proteins, including PP1 regulatory subunit Sds22 in SMAPP1-treated T cells. Docking analysis identified a PP1 binding site for SMAPP1 located within the C-terminal binding pocket of PP1. CONCLUSION: We identified a novel class of PP1-targeting compounds that reactivate latent HIV-1 provirus by targeting PP1, increasing CDK9 phosphorylation and enhancing HIV transcription. This compound represents a novel candidate for anti-HIV-1 therapeutics aiming at eradication of latent HIV-1 reservoirs.

Human papillomavirus type 16 E7 oncoprotein upregulates the retinoic acid receptor-beta expression in cervical cancer cell lines and K14E7 transgenic mice.

Persistent infection with high-risk human papillomaviruses is the main etiological factor in cervical cancer (CC). The human papillomavirus type 16 (HPV16) E7 oncoprotein alters several cellular processes, regulating the expression of many genes in order to avoid cell cycle control. Retinoic acid receptor beta (RARB) blocks cell growth, inducing differentiation and apoptosis. This tumor suppressor gene is gradually silenced in late passages of foreskin keratinocytes immortalized with HPV16 and in various tumors, including CC, mainly by epigenetic modifications. We investigated the effect of E7 oncoprotein on RARB gene expression. We found that HPV16 E7 increases RARB mRNA and RAR-beta protein expression both in vitro and in the cervix of young K14E7 transgenic mice. In E7-expressing cells, RARB overexpression is further increased in the presence of the tumor suppressor p53 (TP53) R273C mutant. This effect does not change when either C33-A or E7-expressing C33-A cell line is treated with Trichostatin A, suggesting that E7 enhances RARB expression independently of histone deacetylases inhibition. These findings indicate that RARB overexpression is part of the early molecular events induced by the E7 oncoprotein.

Design, synthesis and biological evaluation of ezrin inhibitors targeting metastatic osteosarcoma.

Respiratory failure due to pulmonary metastasis is the major cause of death for patients with osteosarcoma. However, the molecular basis for metastasis of osteosarcoma is poorly understood. Recently, ezrin, a member of the ERM family of proteins, has been associated with osteosarcoma metastasis to the lungs. The small molecule NSC 668394 was identified to bind to ezrin, inhibit in vitro and in vivo cell migration, invasion, and metastatic colony survival. Reported herein are the design and synthesis of analogues of NSC 668394, and subsequent functional ezrin inhibition studies. The binding affinity was characterized by surface plasmon resonance technique. Cell migration and invasion activity was determined by electrical cell impedance methodology. Optimization of a series of heterocyclic-dione analogues led to the discovery of compounds 21k and 21m as potential novel antimetastatic agents.

Valosin containing protein (VCP/p97) is a novel substrate for the protein tyrosine phosphatase PTPL1.

Identification of Protein Tyrosine Phosphatase (PTP) substrates is critical in understanding cellular role in normal cells as well as cancer cells. We have previously shown that reduction of PTPL1 protein levels in Ewings sarcoma (ES) inhibit cell growth and tumorigenesis. Therefore, we sought to identify novel PTPL1 substrates that may be important for tumorigenesis. In this current work, we demonstrated that mouse embryonic fibroblasts without PTPL1 catalytic activity fail to form foci when transfected with oncogenes. We proved that catalytic activity of PTPL1 is important for ES cell growth. Using a substrate-trapping mutant of PTPL1 we identified putative PTPL1 substrates by mass-spectrometry. One of these putative substrates was characterized as Valosin Containing Protein (VCP/p97). Using multiple biochemical assays we validated VCP as a novel substrate of PTPL1. We also provide evidence that tyrosine phosphorylation of VCP might be important for its midbody localization during cytokinesis. In conclusion, our work identifies VCP as a new substrate for PTPL1, which may be important in cellular transformation. Our investigation link an oncogenic transcription factor EWS-FLI1, with a key transcriptional target protein tyrosine phosphatase PTPL1, and its substrate VCP. Given our observation that PTPL1 catalytic activity is important for cell transformation, our results may also suggest that VCP regulation by PTPL1 might be important for tumorigenesis.

Loss of SS18-SSX1 inhibits viability and induces apoptosis in synovial sarcoma.

BACKGROUND: Most synovial sarcomas contain a chromosomal translocation t(X;18), which results in the formation of an oncoprotein SS18-SSX critical to the viability of synovial sarcoma. QUESTIONS/PURPOSES: We (1) established and characterized three novel synovial sarcoma cell lines and asked (2) whether inhibition of SS18-SSX1 decreases cell viability in these cell lines; and (3) whether reduction in viability after SS18-SSX1 knockdown is caused by apoptosis. After identifying a specific posttranscriptional splice variant in our cell lines, we asked (4) whether this provides a survival benefit in synovial sarcoma. METHODS: Cells lines were characterized. SS18-SSX1 knockdown was achieved using a shRNA system. Cell viability was assessed by WST-1 analysis and apoptosis examined by caspase-3 activity. RESULTS: We confirmed the SS18-SSX1 translocation in all cell lines and identified a consistent splicing variant. We achieved successful knockdown of SS18-SSX1 and with this saw a significant reduction in cell viability. Decreased viability was a result of increased apoptosis. Reintroduction of the exon 8 sequence into cells reduced cell viability in all cell lines. CONCLUSIONS: We confirmed the presence of the SS18-SSX1 translocation in our cell lines and its importance in the survival of synovial sarcoma. We have also demonstrated that reduction in cell viability is related to an increase in apoptosis. In addition, we have identified a potential mediator of SS18-SSX function in exon 8. CLINICAL RELEVANCE: SS18-SSX represents a tumor-specific target in synovial sarcoma. Exploitation of SS18-SSX and its protein partners will allow us to develop potent tumor-specific therapeutic agents.

The PAX Genes: Roles in Development, Cancer, and Other Diseases.

Since their 1986 discovery in Drosophila, Paired box (PAX) genes have been shown to play major roles in the early development of the eye, muscle, skeleton, kidney, and other organs. Consistent with their roles as master regulators of tissue formation, the PAX family members are evolutionarily conserved, regulate large transcriptional networks, and in turn can be regulated by a variety of mechanisms. Losses or mutations in these genes can result in developmental disorders or cancers. The precise mechanisms by which PAX genes control disease pathogenesis are well understood in some cases, but much remains to be explored. A deeper understanding of the biology of these genes, therefore, has the potential to aid in the improvement of disease diagnosis and the development of new treatments.

Design, synthesis and biological evaluation of Nucleosidic CD99 inhibitors that selectively reduce Ewing sarcoma viability.

Ewing Sarcoma (ES) is a cancer of bone and soft tissues affecting mostly children and young adults. Aggressive progression and poor prognosis of this malignancy call for novel and targeted treatments. CD99 is a transmembrane protein that is abundantly expressed on ES cells and is a diagnostic marker for the disease. ES cells are selectively sensitive to CD99 inhibition compared to most normal cells and other tumors. Therefore, CD99 is a good molecular target for ES treatment. Clofarabine and cladribine are two FDA approved drugs that are administered for their inhibitory acts on DNA synthesis to treat relapsed or refractory acute lymphoblastic and myeloid leukemia. They have also been shown to directly bind to CD99 and inhibit ES growth through a distinct mechanism. In the current study, we designed, synthesized and tested new ES specific derivatives of both drugs that would continue to target CD99 but with expected reduction in cellular membrane permeability and rendered unsuitable for inhibiting DNA synthesis. By using commercially available clofarabine and cladribine purine nucleoside analogs, we modified the primary alcohol moiety at the deoxyribose C-5' terminal site to suppress phosphorylation and thus inhibition of subsequent DNA synthesis pathways. In addition, we incorporated a variety of polar groups in the ribose and purine rings to reduce membrane permeability and investigated the effects of configurational changes in the sugar moiety. Among 26 new derivatives, we identified two compounds, BK50164 and BK60106, that cause cell death specifically in ES primarily due to inhibition of CD99 but not via inhibition of DNA synthesis. These findings provide a road map for the future development selective CD99 inhibitors for targeted treatment of ES.

Piperacetazine Directly Binds to the PAX3::FOXO1 Fusion Protein and Inhibits Its Transcriptional Activity.

The tumor-specific chromosomal translocation product, PAX3::FOXO1, is an aberrant fusion protein that plays a key role for oncogenesis in the alveolar subtype of rhabdomyosarcoma (RMS). PAX3::FOXO1 represents a validated molecular target for alveolar RMS and successful inhibition of its oncogenic activity is likely to have significant clinical applications. Even though several PAX3::FOXO1 function-based screening studies have been successfully completed, a directly binding small-molecule inhibitor of PAX3::FOXO1 has not been reported. Therefore, we screened small-molecule libraries to identify compounds that were capable of directly binding to PAX3::FOXO1 protein using surface plasmon resonance technology. Compounds that directly bound to PAX3::FOXO1 were further evaluated in secondary transcriptional activation assays. We discovered that piperacetazine can directly bind to PAX3::FOXO1 protein and inhibit fusion protein-derived transcription in multiple alveolar RMS cell lines. Piperacetazine inhibited anchorage-independent growth of fusion-positive alveolar RMS cells but not embryonal RMS cells. On the basis of our findings, piperacetazine is a molecular scaffold upon which derivatives could be developed as specific inhibitors of PAX3::FOXO1. These novel inhibitors could potentially be evaluated in future clinical trials for recurrent or metastatic alveolar RMS as novel targeted therapy options. SIGNIFICANCE: RMS is a malignant soft-tissue tumor mainly affecting the pediatric population. A subgroup of RMS with worse prognosis harbors a unique chromosomal translocation creating an oncogenic fusion protein, PAX3::FOXO1. We identified piperacetazine as a direct inhibitor of PAX3::FOXO1, which may provide a scaffold for designing RMS-specific targeted therapy.

Lymphocyte antigen 6K signaling to aurora kinase promotes advancement of the cell cycle and the growth of cancer cells, which is inhibited by LY6K-NSC243928 interaction.

Lymphocyte antigen 6K (LY6K) is a small GPI-linked protein that is normally expressed in testes. Increased expression of LY6K is significantly associated with poor survival outcomes in many solid cancers, including cancers of the breast, ovary, gastrointestinal tract, head and neck, brain, bladder, and lung. LY6K is required for ERK-AKT and TGF-ß pathways in cancer cells and is required for in vivo tumor growth. In this report, we describe a novel role for LY6K in mitosis and cytokinesis through aurora B kinase and its substrate histone H3 signaling axis. Further, we describe the structural basis of the molecular interaction of small molecule NSC243928 with LY6K protein and the disruption of LY6K-aurora B signaling in cell cycle progression due to LY6K-NSC243928 interaction. Overall, disruption of LY6K function via NSC243928 led to failed cytokinesis, multinucleated cells, DNA damage, senescence, and apoptosis of cancer cells. LY6K is not required for vital organ function, thus inhibition of LY6K signaling is an ideal therapeutic approach for hard-to-treat cancers that lack targeted therapy such as triple-negative breast cancer.